Hindawi Publishing Corporation Journal of Drug Delivery Volume 2015, Article ID 790480, 8 pages http://dx.doi.org/10.1155/2015/790480
Research Article Dosing-Time Dependent Effects of Sodium Nitroprusside on Cerebral, Renal, and Hepatic Catalase Activity in Mice Mamane Sani,1,2 Hichem Sebai,3 Roberto Refinetti,2 Mohan Mondal,4 Néziha Ghanem-Boughanmi,3 Naceur A. Boughattas,5 and Mossadok Ben-Attia6 1
UMR Biosurveillance et Toxicologie Environnementale, D´epartement de Biologie, Facult´e des Sciences et Techniques de Maradi, 465 Maradi, Niger 2 Circadian Rhythm Laboratory, Boise State University, 1910 University Drive, Boise, ID 83725, USA 3 UR Ethnobotanie et Stress Oxydant, D´epartement des Sciences de la Vie, Facult´e des Sciences de Bizerte, 7021 Zarzouna, Tunisia 4 National Dairy Research Institute, Eastern Regional Station, A-12, Kalyani,West Bengal 741235, India 5 Laboratoire de Pharmacologie, Facult´e de M´edecine, 5019 Monastir, Tunisia 6 Laboratoire de Biosurveillance de l’Environnement, Facult´e des Sciences de Bizerte, 7021 Zarzouna, Tunisia Correspondence should be addressed to Mamane Sani; [email protected] Received 2 December 2014; Revised 9 February 2015; Accepted 19 February 2015 Academic Editor: A. Fadda Copyright © 2015 Mamane Sani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To investigate the time dependence of sodium nitroprusside- (NPS-) induced oxidative effects, the authors study the variation of the antioxidant enzyme CAT activity in various tissues after the administration of a single 2.5 mg/kg dose of SNP or sodium chloride (NaCl 0.9%). For each of the two dosing times (1 and 13 hours after light onset, HALO, which correspond to the beginning of diurnal rest span and of nocturnal activity span of mice, resp.), brain, kidney, and liver tissues were excised from animals at 0, 1, 3, 6, 9, 12, 24, and 36 h following the drug administration and CAT activity was assayed. The results suggest that SNP-induced stimulation of CAT activity is greater in all three tissues when the drug is administered at 1 HALO than at 13 HALO. Two-way ANOVA revealed that CAT activity significantly (𝑃 < 0.004) varied as a function of the sampling time but not of the treatment in all three tissues. Moreover, a statistically significant (𝑃 < 0.004) interaction between the organ sampling-time and the SNP treatment was revealed in kidney regardless of the dosing time, whereas a highly significant (𝑃 < 0.0002) interaction was validated in liver only in animals injected at 13 HALO.
1. Introduction The use of SNP as an antihypertensive agent [1–4] in a growing list of clinical conditions has been associated with cyanide- (CN− -) induced toxicity [5, 6]. Moreover, previous reports revealed that besides these released CN− ions, other metabolites as nitric monoxide (NO) [7, 8] may also contribute to the toxicity of this drug trough generation of reactive oxygen species (ROS) such as superoxide ion (O2 ∙− ) [5] and hydrogen peroxide (H2 O2 ) [9]. As for many other drugs, side toxic effects of SNP have been reported both in experimental [10–13] and in clinical [14, 15] designs. Indeed, it has been reported that, within minutes of infusion, SNP decomposes into metabolites that are pharmacologically inactive
but toxicologically important [16]. Thus, one molecule of SNP is metabolized by combination with haemoglobin to produce one molecule of cyanmethaemoglobin and four CN ions [17]. Despite this, there have been few reported cases of CN− toxicity following the therapeutic administration of SNP [18–20]. It is well established that the toxic free CN− can be converted in vivo into the much less toxic thiocyanate (SCN) by a ubiquitous enzyme rhodanese [21] that is present in various tissues [22–25] of all living organisms, from bacteria to humans [26–28]. SNP-induced oxidative damage has also been reported [13, 29]. This phenomenon occurs when there is an impairment of the balance between pro- and antioxidant systems. It is well known that SNP-induced oxidative effects are related to the release of NO that might be potentially toxic
2 [7, 8]. Previous studies reported that high levels of NO may interact with ROS or other molecules to generate many more ROS that may induce lipid peroxidation (LPO) in various tissues [30, 31], including brain, kidney, and liver. Moreover, organisms have a wide spectrum of mechanisms to defend against the potential oxidative damage from ROS formation, including antioxidant molecules that directly inactivate ROS and enzymes that metabolically convert toxic compounds to forms that are readily excreted. Among these antioxidant enzymes, CAT seems to play an important role by catalyzing the rapid degradation [32, 33] of H2 O2 to water (H2 O) and oxygen (O2 ). Since there is previous evidence that ROS are important in initiating several pathophysiological processes, especially ischemic reperfusion injury [34, 35], the role of antioxidant enzymes in health and disease is under increasing investigation. Because much of this damage occurs within cells, the antioxidant activity in tissue is considered to be more relevant than that in plasma [36]. Thus, deficiencies compromising the capacity to detoxify oxidant molecules such as H2 O2 and O2 radicals result in oxidant-induced denaturation of intracellular molecules and premature destruction of target cells. Other enzymopathies that might compromise intracellular reductive capacity have also been described; they include abnormalities involving glutathione peroxidase (GPx) and glutathione reductase (GSR) activity [37, 38]. Therefore, any reduced capacity to deal with oxidative stress might involve diminished activity in various antioxidant enzymes or reflect a diminished reserve in the reductive capacity in mutant cells. Although it is clear that antioxidants play an important role in protecting cells, knowledge of molecular mechanisms that regulate their expression is limited. Expression of various genes encoding antioxidant enzymes is partly mediated by a cis-active DNA element designated the antioxidant response element (ARE) [39]. Thus, it is believed that the increase in cell sensitivity results from decreased expression of oxidative-stress response genes. Moreover, the report on downregulation of glutathione-S-transferase (GST) and CAT genes in mutant cells provides a plausible explanation for a molecular basis of the observed sensitivity to oxidant compounds [40]. Since it has been previously reported that CAT plays an essential role in the detoxification of H2 O2 -derived radical species in cells [41–43], a reduction in that enzyme’s activity would be an important factor in the sensitivity of tissues to oxidative stress. Nevertheless, it is likely that expression of a broad set of other enzymes is also affected. Other authors revealed that low concentrations of SNP had a prominent effect in producing oxidative damage in platelets in comparison to rat brain tissue [44]. Similarly, we recently revealed that a neurotoxic 2.5 mg/kg dose of SNP induced LPO in brain, kidney, and liver tissues [13], but not in erythrocytes [45]. Therefore, any variation of oxidative damage among organs might be related to their respective sensitivity and/or antioxidant system efficiency. For that and since we previously found that these three tissues showed different sensitivity to SNPinduced LPO [13], the potentiality and efficiency of their antioxidant systems deserve to be investigated. Hence, we were interested in examining the effects of SNP, a well-known NO donor, on an antioxidant enzyme CAT activity. Since
Journal of Drug Delivery SNP also varies in potency and/or toxicity according to the administration time [11–13, 45], the present study describes the variation of CAT activity in mouse brain, kidney, and liver after i.p. administration of a 2.5 mg/kg dose at two circadian times (1 and 13 HALO).
2. Materials and Methods 2.1. Animals and Housing. Male Swiss albino mice (≈25 g body weight) aged 7–9 weeks (SIPHAT, 2013 FoundoukChoucha, Tunisia) were used. They were acclimated for at least 3 weeks prior to and during experiments [46] in two-air conditioned rooms specially designed for chronobiological investigations by having an inverted light regimen to explore several circadian stages during the usual diurnal work span. In one room, the lights were on from 07:00 to 19:00 h; in the other room, the lights were on from 19:00 to 07:00 h (D : L 12 : 12; reversed lighting regimen). Thus, animals were synchronized with an alternating 12 h light (L)/12 h dark (D) cycle. The room temperature was maintained at 22 ± 2∘ C and the relative humidity was about 50–60%. During all experiments, a standard diet (Purina Rat Chow; SICO, Sfax 3000, Tunisia) and water were provided ad libitum. Animals were randomly divided into four different and comparable groups of 48 mice each at one of the two dosing times 1 and 13 HALO (Table 1). All experiments were performed according to the guidelines of care and use of laboratory animals. 2.2. Drug and Animal Treatment. SNP was kindly supplied by the National Laboratory of Drug Control (1006 Tunis, Tunisia) in the hydrosoluble form (Na2 [Fe(CN)5 NO]⋅2H2 O). Reagents KH2 PO4 , K2 HPO4 , and H2 O2 (110 Vol) stock solutions were obtained from Sigma Aldrich s.r.l. (Milano, Italy) and Merck (Darmstadt, Germany) and were of the highest commercial grade available. Substrate solutions were prepared with distilled water immediately before use. Based on our previous experience with SNP in chronotoxicological studies, in adult mice, neurotoxic effects of SNP were triggered with doses ranging from 2.5 to 5 mg/kg-a median toxic dose TD50 (dose inducing 50% motor-inco-ordination) equal to 3.6 ± 0.5 mg/kg. Since oxidative effects of SNP seem to be related to its neurotoxicity, the lowest neurotoxic SNP dose (of 2.5 mg/kg) was used to investigate SNP-induced oxidative effects. SNP was freshly prepared on each dosing time of the study by adding an adequate volume of sterile distilled water to obtain the desired concentration. For animal experiments, the particular recommendations and approval of protocols were obtained. A single 2.5 mg/kg dose of SNP was administrated to mice by i.p. route in a fixed fluid volume (10 mL/kg b.w.) at each of the two circadian times (1 and 13 HALO). Each circadian time involved different but comparable subgroups of mice (𝑛 = 6) corresponding to animals sacrificed by decapitation at 0, 1, 3, 6, 9, 12, 24, and 36 h after injection. Thereafter, brain, kidney, and liver tissues were quickly removed and individually categorized with respect to tissue, dosing-time, and sampling-time and then stored frozen at −84∘ C until assayed.
Journal of Drug Delivery
3
Table 1: Main characteristics of the study investigating chronotoxicity of SNP in male Swiss albino mice. Drugs NaCl solution (control) SNP NaCl solution (control) SNP
Doses 0.9% 2.5 mg/kg 0.9% 2.5 mg/kg
Number of mice 48 48 48 48
Dosing-time (HALO)
Toxicity variable
Time-of-sampling (HALO)
1
CAT
0, 1, 3, 6, 9, 12, 24, 36
13
CAT
0, 1, 3, 6, 9, 12, 24, 36
500
400
Cerebral CAT activity (𝜇mol H2 O2 /min/g tissue)
Cerebral CAT activity (𝜇mol H2 O2 /min/g tissue)
500 ∗
300 200 100 0
400 300 200 100 0
0
1
3 6 9 12 Time after injection (hours)
24
36
Control NPS
0
1
3 6 9 12 Time after injection (hours)
24
36
Control NPS (a)
(b)
Figure 1: Time course of CAT activity in the brain of mice treated by i.p. route with SNP (2.5 mg/kg) or NaCl (0.9%). (a) Mice were treated with SNP or NaCl at 1 HALO. (b) Mice were treated with SNP or NaCl at 13 HALO. Data are expressed as mean ± SEM values from six different experiments in quadruplicate. The black bar corresponds to the dark period. Unpaired Student’s t-test revealed statistically significance: ∗ 𝑃 < 0.01. Two-way ANOVA: time of sampling ((a) 𝐹0.05(7,88) = 4.8; 𝑃 < 0.004; (b) 𝐹0.05(7,88) = 5.9; 𝑃 < 0.002); treatment ((a) NS; (b) NS); time-treatment interaction ((a) NS; (b) NS).
2.3. Assay Procedure of CAT Activity. For each animal, whole brain, kidneys, and a portion of liver were separately homogenized (5%, w/v) in 20 vol. cold phosphate buffer (10 mM KH2 PO4 –10 mM K2 HPO4 , pH 7.0) using a Petri dish maintained in finely crushed ice. CAT activity was determined at room temperature (26–28∘ C) as described by Claiborne [33]. This method was previously used by the authors to explore temporal variations of CAT activity in brain, kidney, and liver of mice in nonstress conditions [47]. Prior to the measurement of enzyme activity, we demonstrated that the enzyme followed accepted chemical principles by determining the rates of enzyme activity. The rate (𝑅 = 1/time) of CAT activity corresponds to the inverse of length of time (in minutes) for O2 gas formation (visibly apparent as bubbling) after H2 O2 decomposition. We noticed a linear relationship between activity and enzyme concentration. However, according to day, the CAT activity went linearly down by a very small amount. The reaction mixtures (1.95 mL) consisted of 10 mM solution buffer, 100 mM H2 O2 , and a sample. The reaction was initiated by the addition of H2 O2 and absorbance changes were measured at 240 nm. One unit of CAT activity is defined as the amount of enzyme that decomposes 1 mmol H2 O2 per minute. All the results were given as 𝜇mol H2 O2 /min/g tissue. In addition, we assessed the precision of measurements by repeated assay of pools of homogenates that correspond to each tissue. The mean imprecision (coefficient of variation: CV) for within-sample repeatability (intra-assay, 𝑛 = 4) and sample-to-sample reproducibility (interassay, 𝑛 = 6) was
calculated to test whether the variance or the relative SD (CV) was relatively constant between the three studied tissues. The mean CV within-assay was 6.9%, 7.1%, and 8.1% for liver, kidney, and brain, respectively. The mean CV among-assay (inter-assay) was 13.8% for liver, 14.3% for kidney, and 15.2% for brain. 2.4. Statistical Analysis. In the current study, mean and standard error of the mean (S.E.M.) were computed for each sampling-time-point and pertinent histograms were drawn for each circadian dosing-time. The Unpaired Student’s t-test (InStat for MacIntosh, GraphPad Software, San Diego, CA, USA) was used to compare treatment and control groups at designated sampling-times. Two-way analysis of variance (ANOVA) was used to test the significance of differences in CAT activity from one sampling-time to the next by examining the interaction between sampling-time and treatment on the activity levels. Difference is considered statistically significant with a 𝑃 value of 0.05).
DF 7 1 7
Brain 𝐹 4.8 10.4 10.7
Kidney 𝑃
Research Article Dosing-Time Dependent Effects of Sodium Nitroprusside on Cerebral, Renal, and Hepatic Catalase Activity in Mice Mamane Sani,1,2 Hichem Sebai,3 Roberto Refinetti,2 Mohan Mondal,4 Néziha Ghanem-Boughanmi,3 Naceur A. Boughattas,5 and Mossadok Ben-Attia6 1
UMR Biosurveillance et Toxicologie Environnementale, D´epartement de Biologie, Facult´e des Sciences et Techniques de Maradi, 465 Maradi, Niger 2 Circadian Rhythm Laboratory, Boise State University, 1910 University Drive, Boise, ID 83725, USA 3 UR Ethnobotanie et Stress Oxydant, D´epartement des Sciences de la Vie, Facult´e des Sciences de Bizerte, 7021 Zarzouna, Tunisia 4 National Dairy Research Institute, Eastern Regional Station, A-12, Kalyani,West Bengal 741235, India 5 Laboratoire de Pharmacologie, Facult´e de M´edecine, 5019 Monastir, Tunisia 6 Laboratoire de Biosurveillance de l’Environnement, Facult´e des Sciences de Bizerte, 7021 Zarzouna, Tunisia Correspondence should be addressed to Mamane Sani; [email protected] Received 2 December 2014; Revised 9 February 2015; Accepted 19 February 2015 Academic Editor: A. Fadda Copyright © 2015 Mamane Sani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To investigate the time dependence of sodium nitroprusside- (NPS-) induced oxidative effects, the authors study the variation of the antioxidant enzyme CAT activity in various tissues after the administration of a single 2.5 mg/kg dose of SNP or sodium chloride (NaCl 0.9%). For each of the two dosing times (1 and 13 hours after light onset, HALO, which correspond to the beginning of diurnal rest span and of nocturnal activity span of mice, resp.), brain, kidney, and liver tissues were excised from animals at 0, 1, 3, 6, 9, 12, 24, and 36 h following the drug administration and CAT activity was assayed. The results suggest that SNP-induced stimulation of CAT activity is greater in all three tissues when the drug is administered at 1 HALO than at 13 HALO. Two-way ANOVA revealed that CAT activity significantly (𝑃 < 0.004) varied as a function of the sampling time but not of the treatment in all three tissues. Moreover, a statistically significant (𝑃 < 0.004) interaction between the organ sampling-time and the SNP treatment was revealed in kidney regardless of the dosing time, whereas a highly significant (𝑃 < 0.0002) interaction was validated in liver only in animals injected at 13 HALO.
1. Introduction The use of SNP as an antihypertensive agent [1–4] in a growing list of clinical conditions has been associated with cyanide- (CN− -) induced toxicity [5, 6]. Moreover, previous reports revealed that besides these released CN− ions, other metabolites as nitric monoxide (NO) [7, 8] may also contribute to the toxicity of this drug trough generation of reactive oxygen species (ROS) such as superoxide ion (O2 ∙− ) [5] and hydrogen peroxide (H2 O2 ) [9]. As for many other drugs, side toxic effects of SNP have been reported both in experimental [10–13] and in clinical [14, 15] designs. Indeed, it has been reported that, within minutes of infusion, SNP decomposes into metabolites that are pharmacologically inactive
but toxicologically important [16]. Thus, one molecule of SNP is metabolized by combination with haemoglobin to produce one molecule of cyanmethaemoglobin and four CN ions [17]. Despite this, there have been few reported cases of CN− toxicity following the therapeutic administration of SNP [18–20]. It is well established that the toxic free CN− can be converted in vivo into the much less toxic thiocyanate (SCN) by a ubiquitous enzyme rhodanese [21] that is present in various tissues [22–25] of all living organisms, from bacteria to humans [26–28]. SNP-induced oxidative damage has also been reported [13, 29]. This phenomenon occurs when there is an impairment of the balance between pro- and antioxidant systems. It is well known that SNP-induced oxidative effects are related to the release of NO that might be potentially toxic
2 [7, 8]. Previous studies reported that high levels of NO may interact with ROS or other molecules to generate many more ROS that may induce lipid peroxidation (LPO) in various tissues [30, 31], including brain, kidney, and liver. Moreover, organisms have a wide spectrum of mechanisms to defend against the potential oxidative damage from ROS formation, including antioxidant molecules that directly inactivate ROS and enzymes that metabolically convert toxic compounds to forms that are readily excreted. Among these antioxidant enzymes, CAT seems to play an important role by catalyzing the rapid degradation [32, 33] of H2 O2 to water (H2 O) and oxygen (O2 ). Since there is previous evidence that ROS are important in initiating several pathophysiological processes, especially ischemic reperfusion injury [34, 35], the role of antioxidant enzymes in health and disease is under increasing investigation. Because much of this damage occurs within cells, the antioxidant activity in tissue is considered to be more relevant than that in plasma [36]. Thus, deficiencies compromising the capacity to detoxify oxidant molecules such as H2 O2 and O2 radicals result in oxidant-induced denaturation of intracellular molecules and premature destruction of target cells. Other enzymopathies that might compromise intracellular reductive capacity have also been described; they include abnormalities involving glutathione peroxidase (GPx) and glutathione reductase (GSR) activity [37, 38]. Therefore, any reduced capacity to deal with oxidative stress might involve diminished activity in various antioxidant enzymes or reflect a diminished reserve in the reductive capacity in mutant cells. Although it is clear that antioxidants play an important role in protecting cells, knowledge of molecular mechanisms that regulate their expression is limited. Expression of various genes encoding antioxidant enzymes is partly mediated by a cis-active DNA element designated the antioxidant response element (ARE) [39]. Thus, it is believed that the increase in cell sensitivity results from decreased expression of oxidative-stress response genes. Moreover, the report on downregulation of glutathione-S-transferase (GST) and CAT genes in mutant cells provides a plausible explanation for a molecular basis of the observed sensitivity to oxidant compounds [40]. Since it has been previously reported that CAT plays an essential role in the detoxification of H2 O2 -derived radical species in cells [41–43], a reduction in that enzyme’s activity would be an important factor in the sensitivity of tissues to oxidative stress. Nevertheless, it is likely that expression of a broad set of other enzymes is also affected. Other authors revealed that low concentrations of SNP had a prominent effect in producing oxidative damage in platelets in comparison to rat brain tissue [44]. Similarly, we recently revealed that a neurotoxic 2.5 mg/kg dose of SNP induced LPO in brain, kidney, and liver tissues [13], but not in erythrocytes [45]. Therefore, any variation of oxidative damage among organs might be related to their respective sensitivity and/or antioxidant system efficiency. For that and since we previously found that these three tissues showed different sensitivity to SNPinduced LPO [13], the potentiality and efficiency of their antioxidant systems deserve to be investigated. Hence, we were interested in examining the effects of SNP, a well-known NO donor, on an antioxidant enzyme CAT activity. Since
Journal of Drug Delivery SNP also varies in potency and/or toxicity according to the administration time [11–13, 45], the present study describes the variation of CAT activity in mouse brain, kidney, and liver after i.p. administration of a 2.5 mg/kg dose at two circadian times (1 and 13 HALO).
2. Materials and Methods 2.1. Animals and Housing. Male Swiss albino mice (≈25 g body weight) aged 7–9 weeks (SIPHAT, 2013 FoundoukChoucha, Tunisia) were used. They were acclimated for at least 3 weeks prior to and during experiments [46] in two-air conditioned rooms specially designed for chronobiological investigations by having an inverted light regimen to explore several circadian stages during the usual diurnal work span. In one room, the lights were on from 07:00 to 19:00 h; in the other room, the lights were on from 19:00 to 07:00 h (D : L 12 : 12; reversed lighting regimen). Thus, animals were synchronized with an alternating 12 h light (L)/12 h dark (D) cycle. The room temperature was maintained at 22 ± 2∘ C and the relative humidity was about 50–60%. During all experiments, a standard diet (Purina Rat Chow; SICO, Sfax 3000, Tunisia) and water were provided ad libitum. Animals were randomly divided into four different and comparable groups of 48 mice each at one of the two dosing times 1 and 13 HALO (Table 1). All experiments were performed according to the guidelines of care and use of laboratory animals. 2.2. Drug and Animal Treatment. SNP was kindly supplied by the National Laboratory of Drug Control (1006 Tunis, Tunisia) in the hydrosoluble form (Na2 [Fe(CN)5 NO]⋅2H2 O). Reagents KH2 PO4 , K2 HPO4 , and H2 O2 (110 Vol) stock solutions were obtained from Sigma Aldrich s.r.l. (Milano, Italy) and Merck (Darmstadt, Germany) and were of the highest commercial grade available. Substrate solutions were prepared with distilled water immediately before use. Based on our previous experience with SNP in chronotoxicological studies, in adult mice, neurotoxic effects of SNP were triggered with doses ranging from 2.5 to 5 mg/kg-a median toxic dose TD50 (dose inducing 50% motor-inco-ordination) equal to 3.6 ± 0.5 mg/kg. Since oxidative effects of SNP seem to be related to its neurotoxicity, the lowest neurotoxic SNP dose (of 2.5 mg/kg) was used to investigate SNP-induced oxidative effects. SNP was freshly prepared on each dosing time of the study by adding an adequate volume of sterile distilled water to obtain the desired concentration. For animal experiments, the particular recommendations and approval of protocols were obtained. A single 2.5 mg/kg dose of SNP was administrated to mice by i.p. route in a fixed fluid volume (10 mL/kg b.w.) at each of the two circadian times (1 and 13 HALO). Each circadian time involved different but comparable subgroups of mice (𝑛 = 6) corresponding to animals sacrificed by decapitation at 0, 1, 3, 6, 9, 12, 24, and 36 h after injection. Thereafter, brain, kidney, and liver tissues were quickly removed and individually categorized with respect to tissue, dosing-time, and sampling-time and then stored frozen at −84∘ C until assayed.
Journal of Drug Delivery
3
Table 1: Main characteristics of the study investigating chronotoxicity of SNP in male Swiss albino mice. Drugs NaCl solution (control) SNP NaCl solution (control) SNP
Doses 0.9% 2.5 mg/kg 0.9% 2.5 mg/kg
Number of mice 48 48 48 48
Dosing-time (HALO)
Toxicity variable
Time-of-sampling (HALO)
1
CAT
0, 1, 3, 6, 9, 12, 24, 36
13
CAT
0, 1, 3, 6, 9, 12, 24, 36
500
400
Cerebral CAT activity (𝜇mol H2 O2 /min/g tissue)
Cerebral CAT activity (𝜇mol H2 O2 /min/g tissue)
500 ∗
300 200 100 0
400 300 200 100 0
0
1
3 6 9 12 Time after injection (hours)
24
36
Control NPS
0
1
3 6 9 12 Time after injection (hours)
24
36
Control NPS (a)
(b)
Figure 1: Time course of CAT activity in the brain of mice treated by i.p. route with SNP (2.5 mg/kg) or NaCl (0.9%). (a) Mice were treated with SNP or NaCl at 1 HALO. (b) Mice were treated with SNP or NaCl at 13 HALO. Data are expressed as mean ± SEM values from six different experiments in quadruplicate. The black bar corresponds to the dark period. Unpaired Student’s t-test revealed statistically significance: ∗ 𝑃 < 0.01. Two-way ANOVA: time of sampling ((a) 𝐹0.05(7,88) = 4.8; 𝑃 < 0.004; (b) 𝐹0.05(7,88) = 5.9; 𝑃 < 0.002); treatment ((a) NS; (b) NS); time-treatment interaction ((a) NS; (b) NS).
2.3. Assay Procedure of CAT Activity. For each animal, whole brain, kidneys, and a portion of liver were separately homogenized (5%, w/v) in 20 vol. cold phosphate buffer (10 mM KH2 PO4 –10 mM K2 HPO4 , pH 7.0) using a Petri dish maintained in finely crushed ice. CAT activity was determined at room temperature (26–28∘ C) as described by Claiborne [33]. This method was previously used by the authors to explore temporal variations of CAT activity in brain, kidney, and liver of mice in nonstress conditions [47]. Prior to the measurement of enzyme activity, we demonstrated that the enzyme followed accepted chemical principles by determining the rates of enzyme activity. The rate (𝑅 = 1/time) of CAT activity corresponds to the inverse of length of time (in minutes) for O2 gas formation (visibly apparent as bubbling) after H2 O2 decomposition. We noticed a linear relationship between activity and enzyme concentration. However, according to day, the CAT activity went linearly down by a very small amount. The reaction mixtures (1.95 mL) consisted of 10 mM solution buffer, 100 mM H2 O2 , and a sample. The reaction was initiated by the addition of H2 O2 and absorbance changes were measured at 240 nm. One unit of CAT activity is defined as the amount of enzyme that decomposes 1 mmol H2 O2 per minute. All the results were given as 𝜇mol H2 O2 /min/g tissue. In addition, we assessed the precision of measurements by repeated assay of pools of homogenates that correspond to each tissue. The mean imprecision (coefficient of variation: CV) for within-sample repeatability (intra-assay, 𝑛 = 4) and sample-to-sample reproducibility (interassay, 𝑛 = 6) was
calculated to test whether the variance or the relative SD (CV) was relatively constant between the three studied tissues. The mean CV within-assay was 6.9%, 7.1%, and 8.1% for liver, kidney, and brain, respectively. The mean CV among-assay (inter-assay) was 13.8% for liver, 14.3% for kidney, and 15.2% for brain. 2.4. Statistical Analysis. In the current study, mean and standard error of the mean (S.E.M.) were computed for each sampling-time-point and pertinent histograms were drawn for each circadian dosing-time. The Unpaired Student’s t-test (InStat for MacIntosh, GraphPad Software, San Diego, CA, USA) was used to compare treatment and control groups at designated sampling-times. Two-way analysis of variance (ANOVA) was used to test the significance of differences in CAT activity from one sampling-time to the next by examining the interaction between sampling-time and treatment on the activity levels. Difference is considered statistically significant with a 𝑃 value of 0.05).
DF 7 1 7
Brain 𝐹 4.8 10.4 10.7
Kidney 𝑃
